Project Summary Deciphering the molecular mechanisms underlying genomic instability and tumorigenesis is the long-term goal of my laboratory. The broad objective, of this proposal, reflects our pursuit to gain a comprehensive understanding of the network involved in DNA repair and to determine how these proteins and pathways intersect, interact, communicate, coordinate, and collaborate for genome maintenance. The short-term goal is to perform detailed mechanistic studies of several DNA damage signaling and repair pathways, which will provide the foundation to achieve our long-term goal of exploiting DNA repair network for cancer therapy. This proposal will define two overlapping DNA damage-signaling pathways that together are essential for cell survival. We and other researchers have constructed an elaborate signaling pathway that acts downstream of H2AX and regulates the recruitment and accumulation of many DNA damage repair proteins at sites of DNA breaks. This H2AX-dependent pathway is composed of H2AX, MDC1, RNF8, and RNF168. However, repair defects observed in H2AX-, MDC1-, RNF8-, or RNF168-deficient cells or mice are mild, raising the possibility that there is an H2AX-independent mechanism involved in the recruitment of these downstream repair proteins. We propose that this H2AX-independent pathway is controlled by NBS1. On the basis of our previous studies and preliminary data presented in this proposal, we hypothesize that the H2AX- and NBS1-dependent pathways are involved in the DNA damage response and are critical for cell survival. We believe that these two pathways have redundant functions, especially in promoting homologous recombination repair. However, they are not completely separate, since they intersect at multiple points. This makes it considerably challenging for us to delineate the functions of these two redundant pathways. It is unknown whether we can elucidate the contribution of a single pathway to the ever-growing network, i.e., can we untangle the network to understand the mechanisms by which different pathways intersect and contribute to biological processes? We will address this question in this application and we will further study the mechanisms by which the H2AX- and NBS1-dependent pathways act together to ensure cell survival and the completion of DNA repair. We propose the following specific aims: 1) determine whether NBS1 acts redundantly with the established H2AX-MDC1-RNF8-RNF168 pathway to ensure cell survival; 2) delineate the NBS1-dependent pathway; and 3) explore the mechanisms underlying cell lethality caused by NBS1 and H2AX co-depletion. These studies will not only allow us to understand the redundant functions of H2AX and NBS1 in vivo but will also reveal ways to investigate the functions of proteins and pathways in today?s complex signaling networks. Moreover, results from these studies will provide the rationale for exploiting DNA repair defect and applying synthetic lethality concept in precision medicine for cancer patients.